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Ongoing projects include the following:

1)ÌýSystems biology of TGF-β signaling in normal and cancer cells
2)ÌýChemical biology and biochemistry of the ubiquitin system

3)ÌýMps1 kinase and spindle checkpoint signaling
4)ÌýCis-regulatory transcriptional regulation

Systems biology ofÌýTGF-β signaling in normal and cancer cells

We aim to understand the role of transforming growth factor-β (TGF-β) signaling in normal and cancer cells. TGF-β is a multi-functional cytokine responsible for regulating growth and differentiation of a wide variety of cell types and tissues. It is a potent inhibitor of normal epithelial cell proliferation and possesses tumor suppressing activity. The majority of human tumors are of epithelial origin and their proliferation is no longer inhibited by TGF-β. This usually arises either from a loss of key signaling molecules in the TGF-β signal transduction pathway (e.g., TGF-β receptors), or from activation of oncogenes. TGF-β can positively regulate expression of many tumor suppressor genes which cause the cell to stop proliferation and yet negatively regulate expression of many proto-oncogenes/oncogenes which promote cell cycle progression. The action of TGF-β in normal epithelial cells is to downshift the engine that drives cell proliferation by shifting the balance of the activity of the oncogene and tumor suppressor gene.

TGF-β signals via two receptor Ser/Thr protein kinases, termed type I and type II TGF-β receptors. The type II receptor phosphorylates and activates the type I receptor, which then phosphorylates Smad2 or Smad3 within their C-terminal Ser-Ser-X-Ser motifs. Once activated, Smad2 or Smad3 associate with the shared signaling molecule Smad4, translocate to the nucleus and in concert with additional transcription factors alters the transcription of a large repertoire of genes. Although several key downstream targets that transduce the TGF-β signals from the cell surface to the nucleus have been identified, it remains to be determined how TGF-β can mediate myriad cellular responses and regulate so many important physiological processes. To address how TGF-β signaling could mediate such diverse effects, we use the two following approaches: 1) Find as yet unidentified molecules that may mediate diverse functions, and 2) understand how the TGF-β network behaves as a system, which may identify how known network components interact to produce unexpected emergent behavior. The principal methods used to achieve these aims include cDNA library screening for genes that mediate TGF-β resistance (Erickson et al., MBC 2009), purification and identification by mass spectrometry of protein complexes associated with well-established signaling transducers (Knuesel et al., 2003, Zhu et al., 2007) and computational modeling of TGF- β/Smad signaling dynamics (Clarke et al., 2006; Clarke et al. 2008; Clarke et al. 2009; Zi et al. 2011).

Chemical biology and biochemistry of the ubiquitin system

Proteins in the cell are subject to diverse covalent modifications that serve to alter their activity, localization, or turnover. Such modifications include ubiquitination and covalent attachment of ubiquitin-like molecules (e.g., SUMO or small ubiquitin-like modifier). We initially became interested in how these modifications regulate cell cycle proteins that could disrupt TGF-β signaling (Liu et al., 2000). We subsequently found further interplay between TGF-β signaling and protein degradation mechanisms (Wang et al., 2004, MacDonald et al., 2004). More recently, we have sought to determine the biochemical mechanisms by which E3 ligases mediate ubiquitination of their substrates (Wang et al., 2004, Ungermannova et al., 2005, Wang et al., 2005; Wang et al. 2006). We continue to pursue these studies using structural and biochemical techniques.Ìý We are mainly interested in two SCF type of ubiquitin ligases (SCFSkp2 and SCFFbx4; Zeng et al. 2010).Ìý More recently we developed high throughput screening assays for identifying small molecule inhibitors of SCF E3 ligase in an effort to curb excessive proteolysis of p27Kip1 which is a tumor suppressor protein perturbed in a variety of human tumors. In addition, studies were done to establish the functional importance of post-translational modifications on the activity of transcription factors. Here, muscle differentiation, which is largely driven through transcriptional regulation, was used as a model system (Riquelme et al., 2006, 2 separate publications).

Mps1 kinase and spindle checkpoint signaling

A hallmark of tumor cells is an inability to control their proliferation, which implies that these cells are capable of bypassing cell cycle checkpoints and regulation by tumor suppressor signaling pathways.Ìý Aberrant chromosome segregation generates aneuploid cells and genome instability, which has been postulated to be a major mechanism for tumorigenesis.Ìý Aneuploidy is primarily caused by errors during mitosis.Ìý In normal cells, correct segregation of chromosomes is ensured by an evolutionarily conserved surveillance signal transduction pathway called the mitotic spindle checkpointÌýÌý TTK/Mps1, a dual specificity protein kinase, has emerged as a master regulator of mitosis and an upstream component of spindle checkpoint signaling pathway.Ìý Our previous work led to identification of Smad proteins as substrates for Mps1 in mitosis (Zhu et al. 2007).Ìý Recently we solved the crystal structure of the Mps1 kinase domain in collaboration with Ming Lei's group (Wang et al. 2008).Ìý We demonstrated that autophosphorylation of Mps1 is a priming mechanism for Mps1 activation and critical for kinetochore targeting (Wang et al. 2008, Xu et al. 2009; Sun et al. 2010; Zhang et al. 2011).Ìý Currently we are interested in understanding how Mps1 kinase is turned on and off in a cell cycle dependent manner and how its activity is perturbed in tumor cells to weaken the spindle checkpoint. Ìý

Cis-regulatory transcriptional regulation

Genome sequencing has revealed the basic molecular blueprint for diverse organisms. How such sequences give rise to the development and physiology of such organisms is of great interest. Many processes are regulated at the transcriptional level, which are mediated by transcription factor proteins that bind specific and distinct DNA sequences in the promoter and enhancer regions of genes. Predicting which transcription factors bind the promoters of genes of interest is crucial for understanding transcriptional regulation. We have developed a suite of computational tools,Ìýcalled GeneACT,for studying global transcriptional regulation (Cheung et al. 2007).Ìý We have used these tools to identify transcription factors important for mediating myogenic differentiation and TGF-β-induced gene responses (Cheung et al., 2007; Barthel et al. 2008).

ÌýZi, Z., D.A. Chapnick, and X. Liu, Dynamics of TGF-beta/Smad Signaling.ÌýFEBS Lett, 586 (2012) 1921–1928.

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Ungermannova, D., S.J. Parker, C.G. Nasveschuk, W. Wang, B. Quade, G. Zhang, R.D. Kuchta, A.J. Phillips, and X. Liu, Largazole and Its Derivatives Selectively Inhibit Ubiquitin Activating Enzyme (E1).ÌýPLoS One, 2012. 7(1): p. e29208.

Ungermannova, D., S.J. Parker, C.G. Nasveschuk, D.A. Chapnick, A.J. Phillips, R.D. Kuchta, and X. Liu, Identification and Mechanistic Studies of a Novel Ubiquitin E1 Inhibitor.ÌýJ Biomol Screen, 2012. 17(4): p. 421-34.

Liu, X. and M. Winey, The Mps1 Family of Protein Kinases.ÌýAnnu Rev Biochem, 2012. 81: p. 561-85.

Zi, Z., Z. Feng, D.A. Chapnick, M. Dahl, D. Deng, E. Klipp, A. Moustakas, and X. Liu, Quantitative Analysis of Transient and Sustained Transforming Growth Factor-Beta Signaling Dynamics.ÌýMol Syst Biol, 2011. 7: p. 492.

Zhang, X., Q. Yin, Y. Ling, Y. Zhang, R. Ma, Q. Ma, C. Cao, H. Zhong, X. Liu, and Q. Xu, Two Lxxll Motifs in the N Terminus of Mps1 Are Required for Mps1 Nuclear Import During G(2)/M Transition and Sustained Spindle Checkpoint Responses.ÌýCell Cycle, 2011. 10(16): p. 2742-50.

He, J., J. Ye, Y. Cai, C. Riquelme, J.O. Liu, X. Liu, A. Han, and L. Chen, Structure of P300 Bound to Mef2 on DNA Reveals a Mechanism of Enhanceosome Assembly.ÌýNucleic Acids Res, 2011. 39(10): p. 4464-74.

Chapnick, D.A., L. Warner, J. Bernet, T. Rao, and X. Liu, Partners in Crime: Tgf-Beta and Mapk Pathways in Cancer Progression.ÌýCell Biosci, 2011. 1(1): p. 42.

Zhong, J., X. Liu, and A. Pandey, Effects of Transmembrane and Juxtamembrane Domains on Proliferative Ability of Tslp Receptor.ÌýMol Immunol, 2010. 47(6): p. 1207-15.

Zeng, Z., W. Wang, Y. Yang, Y. Chen, X. Yang, J.A. Diehl, X. Liu*, and M. Lei*, Structural Basis of Selective Ubiquitination of Trf1 by Scffbx4.ÌýDev Cell, 2010. 18(2): p. 214-25.Ìý * Corresponding authors.

Sun, T., X. Yang, W. Wang, X. Zhang, Q. Xu, S. Zhu, R. Kuchta, G. Chen, and X. Liu, Cellular Abundance of Mps1 and the Role of Its Carboxyl Terminal Tail in Substrate Recruitment.ÌýJ Biol Chem, 2010. 285(49): p. 38730-9.

Clarke, D.C. and X. Liu, Measuring the Absolute Abundance of the Smad Transcription Factors Using Quantitative Immunoblotting.ÌýMethods Mol Biol, 2010. 647: p. 357-76.

Chapnick, D.A. and X. Liu, Analysis of Ligand-Dependent Nuclear Accumulation of Smads in Tgf-Beta Signaling.ÌýMethods Mol Biol, 2010. 647: p. 95-111.

Xu, Q., S. Zhu, W. Wang, X. Zhang, W. Old, N. Ahn, and X. Liu, Regulation of Kinetochore Recruitment of Two Essential Mitotic Spindle Checkpoint Proteins by Mps1 Phosphorylation.ÌýMol Biol Cell, 2009. 20(1): p. 10-20.

Wang, W., Y. Yang, Y. Gao, Q. Xu, F. Wang, S. Zhu, W. Old, K. Resing, N. Ahn, M. Lei*, and X. Liu*, Structural and Mechanistic Insights into Mps1 Kinase Activation.ÌýJ Cell Mol Med, 2009. 13(8B): p. 1679-94. *corresponding authors.

Granovsky, A.E., M.C. Clark, D. McElheny, G. Heil, J. Hong, X. Liu, Y. Kim, G. Joachimiak, A. Joachimiak, S. Koide, and M.R. Rosner, Raf Kinase Inhibitory Protein Function Is Regulated Via a Flexible Pocket and Novel Phosphorylation-Dependent Mechanism.ÌýMol Cell Biol, 2009. 29(5): p. 1306-20.

Erickson, R.A. and X. Liu, Association of V-Erba with Smad4 Disrupts Tgf-Beta Signaling.ÌýMol Biol Cell, 2009. 20(5): p. 1509-19.

Clarke, D.C., M.L. Brown, R.A. Erickson, Y. Shi, and X. Liu, Transforming Growth Factor Beta Depletion Is the Primary Determinant of Smad Signaling Kinetics.ÌýMol Cell Biol, 2009. 29(9): p. 2443-55.

Nasveschuk, C.G., D. Ungermannova, X. Liu, and A.J. Phillips, A Concise Total Synthesis of Largazole, Solution Structure, and Some Preliminary Structure Activity Relationships.ÌýOrg Lett, 2008. 10(16): p. 3595-8.

Guo, X., D.S. Waddell, W. Wang, Z. Wang, N.T. Liberati, S. Yong, X. Liu, and X.F. Wang, Ligand-Dependent Ubiquitination of Smad3 Is Regulated by Casein Kinase 1 Gamma 2, an Inhibitor of Tgf-Beta Signaling.ÌýOncogene, 2008. 27(58): p. 7235-47.

Guo, X., A. Ramirez, D.S. Waddell, Z. Li, X. Liu, and X.F. Wang, Axin and Gsk3- Control Smad3 Protein Stability and Modulate Tgf- Signaling.ÌýGenes Dev, 2008. 22(1): p. 106-20.

Clarke, D.C. and X. Liu, Decoding the Quantitative Nature of Tgf-Beta/Smad Signaling.ÌýTrends Cell Biol, 2008. 18(9): p. 430-42.

Barthel, K.K. and X. Liu, A Transcriptional Enhancer from the Coding Region of Adamts5.ÌýPLoS ONE, 2008. 3(5): p. e2184.

Zhu, S., W. Wang, D.C. Clarke, and X. Liu, Activation of Mps1 Promotes Transforming Growth Factor-Beta-Independent Smad Signaling.ÌýJ Biol Chem, 2007. 282(25): p. 18327-38.

Zhang, L., L. Ding, T.H. Cheung, M.Q. Dong, J. Chen, A.K. Sewell, X. Liu, J.R. Yates, 3rd, and M. Han, Systematic Identification of C. Elegans Mirisc Proteins, Mirnas, and Mrna Targets by Their Interactions with Gw182 Proteins Ain-1 and Ain-2.ÌýMol Cell, 2007. 28(4): p. 598-613.

Cheung, T.H., K.K. Barthel, Y.L. Kwan, and X. Liu, Identifying Pattern-Defined Regulatory Islands in Mammalian Genomes.ÌýProc Natl Acad Sci U S A, 2007. 104(24): p. 10116-21.

Riquelme, C., K.K. Barthel, X.F. Qin, and X. Liu, Ubc9 Expression Is Essential for Myotube Formation in C2c12.ÌýExp Cell Res, 2006. 312(11): p. 2132-41.

Riquelme, C., K.K. Barthel, and X. Liu, Sumo-1 Modification of Mef2a Regulates Its Transcriptional Activity.ÌýJ Cell Mol Med, 2006. 10(1): p. 132-44.

Clarke, D.C., M.D. Betterton, and X. Liu, Systems Theory of Smad Signalling.ÌýSyst Biol (Stevenage), 2006. 153(6): p. 412-24.

Cheung, T.H., Y.L. Kwan, M. Hamady, and X. Liu, Unraveling Transcriptional Control and Cis-Regulatory Codes Using the Software Suite Geneact.ÌýGenome Biol, 2006. 7(10): p. R97.

Wang, W., L. Nacusi, R.J. Sheaff, and X. Liu, Ubiquitination of P21cip1/Waf1 by Scfskp2: Substrate Requirement and Ubiquitination Site Selection.ÌýBiochemistry, 2005. 44(44): p. 14553-64.

Ungermannova, D., Y. Gao, and X. Liu, Ubiquitination of P27kip1 Requires Physical Interaction with Cyclin E and Probable Phosphate Recognition by Skp2.ÌýJ Biol Chem, 2005. 280(34): p. 30301-9.

Knuesel, M., H.T. Cheung, M. Hamady, K.K. Barthel, and X. Liu, A Method of Mapping Protein Sumoylation Sites by Mass Spectrometry Using a Modified Small Ubiquitin-Like Modifier 1 (Sumo-1) and a Computational Program.Mol Cell Proteomics, 2005. 4(10): p. 1626-36.

Kfir, S., M. Ehrlich, A. Goldshmid, X. Liu, Y. Kloog, and Y.I. Henis, Pathway- and Expression Level-Dependent Effects of Oncogenic N-Ras: P27(Kip1) Mislocalization by the Ral-Gef Pathway and Erk-Mediated Interference with Smad Signaling.ÌýMol Cell Biol, 2005. 25(18): p. 8239-50.

Wang, W., D. Ungermannova, J. Jin, J.W. Harper, and X. Liu, Negative Regulation of Scfskp2 Ubiquitin Ligase by Tgf-Beta Signaling.ÌýOncogene, 2004. 23(5): p. 1064-75.

Wang, W., D. Ungermannova, L. Chen, and X. Liu, Molecular and Biochemical Characterization of the Skp2-Cks1 Binding Interface.ÌýJ Biol Chem, 2004. 279(49): p. 51362-9.

Royer, Y., C. Menu, X. Liu, and S.N. Constantinescu, High-Throughput Gateway Bicistronic Retroviral Vectors for Stable Expression in Mammalian Cells: Exploring the Biologic Effects of Stat5 Overexpression.ÌýDNA Cell Biol, 2004. 23(6): p. 355-65.

Macdonald, M., Y. Wan, W. Wang, E. Roberts, T.H. Cheung, R. Erickson, M.T. Knuesel, and X. Liu, Control of Cell Cycle-Dependent Degradation of C-Ski Proto-Oncoprotein by Cdc34.ÌýOncogene, 2004. 23(33): p. 5643-53.

Liang, M., Y.Y. Liang, K. Wrighton, D. Ungermannova, X.P. Wang, F.C. Brunicardi, X. Liu, X.H. Feng, and X. Lin, Ubiquitination and Proteolysis of Cancer-Derived Smad4 Mutants by Scfskp2.ÌýMol Cell Biol, 2004. 24(17): p. 7524-37.

Wang, W., D. Ungermannova, L. Chen, and X. Liu, A Negatively Charged Amino Acid in Skp2 Is Required for Skp2-Cks1 Interaction and Ubiquitination of P27kip1.ÌýJ Biol Chem, 2003. 278(34): p. 32390-6.

Knuesel, M., Y. Wan, Z. Xiao, E. Holinger, N. Lowe, W. Wang, and X. Liu, Identification of Novel Protein-Protein Interactions Using a Versatile Mammalian Tandem Affinity Purification Expression System.ÌýMol Cell Proteomics, 2003. 2(11): p. 1225-33.

Wan, Y., X. Liu, and M.W. Kirschner, The Anaphase-Promoting Complex Mediates Tgf-Beta Signaling by Targeting Snon for Destruction.ÌýMol Cell, 2001. 8(5): p. 1027-39.

Liu, X., Y. Sun, R.A. Weinberg, and H.F. Lodish, Ski/Sno and Tgf-Beta Signaling.ÌýCytokine Growth Factor Rev, 2001. 12(1): p. 1-8.

Fu, M., J. Zhang, X. Zhu, D.E. Myles, T.M. Willson, X. Liu, and Y.E. Chen, Peroxisome Proliferator-Activated Receptor Gamma Inhibits Transforming Growth Factor Beta-Induced Connective Tissue Growth Factor Expression in Human Aortic Smooth Muscle Cells by Interfering with Smad3.ÌýJ Biol Chem, 2001. 276(49): p. 45888-94.

*Blobe, G.C., X. Liu*, S.J. Fang, T. How, and H.F. Lodish, A Novel Mechanism for Regulating Transforming Growth Factor Beta (Tgf-Beta) Signaling. Functional Modulation of Type Iii Tgf-Beta Receptor Expression through Interaction with the Pdz Domain Protein, Gipc.ÌýJ Biol Chem, 2001. 276(43): p. 39608-17. *Equal contributions.

Xiao, Z., X. Liu, and H.F. Lodish, Importin Beta Mediates Nuclear Translocation of Smad 3.ÌýJ Biol Chem, 2000. 275(31): p. 23425-8.

Xiao, Z., X. Liu, Y.I. Henis, and H.F. Lodish, A Distinct Nuclear Localization Signal in the N Terminus of Smad 3 Determines Its Ligand-Induced Nuclear Translocation.ÌýProc Natl Acad Sci U S A, 2000. 97(14): p. 7853-8.

Liu, X., Y. Sun, M. Ehrlich, T. Lu, Y. Kloog, R.A. Weinberg, H.F. Lodish, and Y.I. Henis, Disruption of Tgf-Beta Growth Inhibition by Oncogenic Ras Is Linked to P27kip1 Mislocalization.ÌýOncogene, 2000. 19(51): p. 5926-35.

Liu, X., S.N. Constantinescu, Y. Sun, J.S. Bogan, D. Hirsch, R.A. Weinberg, and H.F. Lodish, Generation of Mammalian Cells Stably Expressing Multiple Genes at Predetermined Levels.ÌýAnal Biochem, 2000. 280(1): p. 20-8.

Wells, R.G., L. Gilboa, Y. Sun, X. Liu, Y.I. Henis, and H.F. Lodish, Transforming Growth Factor-Beta Induces Formation of a Dithiothreitol-Resistant Type I/Type Ii Receptor Complex in Live Cells.ÌýJ Biol Chem, 1999. 274(9): p. 5716-22.

Sun, Y., X. Liu, E. Ng-Eaton, H.F. Lodish, and R.A. Weinberg, Snon and Ski Protooncoproteins Are Rapidly Degraded in Response to Transforming Growth Factor Beta Signaling.ÌýProc Natl Acad Sci U S A, 1999. 96(22): p. 12442-7.

Sun*, Y., X. Liu*, E.N. Eaton, W.S. Lane, H.F. Lodish, and R.A. Weinberg, Interaction of the Ski Oncoprotein with Smad3 Regulates Tgf-Beta Signaling.ÌýMol Cell, 1999. 4(4): p. 499-509.Ìý *Equal contributions.

Constantinescu, S.N., X. Liu, W. Beyer, A. Fallon, S. Shekar, Y.I. Henis, S.O. Smith, and H.F. Lodish, Activation of the Erythropoietin Receptor by the Gp55-P Viral Envelope Protein Is Determined by a Single Amino Acid in Its Transmembrane Domain.ÌýEmbo J, 1999. 18(12): p. 3334-47.

Kuo, J.S., M. Patel, J. Gamse, C. Merzdorf, X. Liu, V. Apekin, and H. Sive, Opl: A Zinc Finger Protein That Regulates Neural Determination and Patterning in Xenopus.ÌýDevelopment, 1998. 125(15): p. 2867-82.

Hua, X., X. Liu, D.O. Ansari, and H.F. Lodish, Synergistic Cooperation of Tfe3 and Smad Proteins in Tgf-Beta-Induced Transcription of the Plasminogen Activator Inhibitor-1 Gene.ÌýGenes Dev, 1998. 12(19): p. 3084-95.

Constantinescu, S.N., H. Wu, X. Liu, W. Beyer, A. Fallon, and H.F. Lodish, The Anemic Friend Virus Gp55 Envelope Protein Induces Erythroid Differentiation in Fetal Liver Colony-Forming Units-Erythroid.ÌýBlood, 1998. 91(4): p. 1163-72.

Liu, X., Y. Sun, S.N. Constantinescu, E. Karam, R.A. Weinberg, and H.F. Lodish, Transforming Growth Factor Beta-Induced Phosphorylation of Smad3 Is Required for Growth Inhibition and Transcriptional Induction in Epithelial Cells.ÌýProc Natl Acad Sci U S A, 1997. 94(20): p. 10669-74.

Johnston, S.D., X. Liu, F. Zuo, T.L. Eisenbraun, S.R. Wiley, R.J. Kraus, and J.E. Mertz, Estrogen-Related Receptor Alpha 1 Functionally Binds as a Monomer to Extended Half-Site Sequences Including Ones Contained within Estrogen-Response Elements.ÌýMol Endocrinol, 1997. 11(3): p. 342-52.

Liu, X. and J.E. Mertz, Sequence of the Polypyrimidine Tract of the 3'-Terminal 3' Splicing Signal Can Affect Intron-Dependent Pre-Mrna Processing in Vivo.ÌýNucleic Acids Res, 1996. 24(9): p. 1765-73.

Liu, X. and J.E. Mertz, Hnrnp L Binds a Cis-Acting Rna Sequence Element That Enables Intron-Dependent Gene Expression.ÌýGenes Dev, 1995. 9(14): p. 1766-80.

Liu, X. and J.E. Mertz, Polyadenylation Site Selection Cannot Occur in Vivo after Excision of the 3'-Terminal Intron.ÌýNucleic Acids Res, 1993. 21(22): p. 5256-63.